1. Field of the Invention
This invention relates to compositions useful for the prevention and treatment of parasitic infections which cause diseases such as malaria, trypanosomiasis, leishmania, schistosomiasis and elephantitis. Useful compositions contain an adenosine or adenosine derivative and, optionally, a deaminase inhibitor. Compositions may be used in vivo to treat patients and also in vitro to inactivate or destroy infectious organisms in cultured cells, in fluids such as blood and blood products, in biological products derived from cells and in foods. This invention also relates to methods for treating or preventing parasitic infections with these compositions.
2. Description of the Background
Parasitism is a normal and ubiquitous part of all aspects of life and has been found to occur in nearly every living organism. A wide variety of organisms parasitize prokaryotics and an even wider variety plants and animals. As a general rule, the more complex the host the greater the number and variety of parasites which are possible and probable.
There is a diverse array of host-parasite relationships found to occur between parasites and hosts. These relationships can be divided into three major groups, mutualism, commensalism and true parasitism. Mutualism, or mutual symbiosis, occurs in those relationships where both the parasite and the host receive some benefit. One example of this type of relationship is found between the protozoan that inhabits the termite gut and the termite. Termites ingest wood and play an important if not essential role in the life cycle of a forest. Termites can perform this task because of the particular flora which inhabit their gut. These microorganisms carry and express the enzymes necessary to break down cellulosic materials, but do not appear to be detrimental to the termite. The benefits to both host and parasite are clear. Another form of parasitism is commensalism where one member of the association receives all or at least most of the benefits of the relationship while the other is neither harmed or benefited. True parasitism is where the parasite inflicts some degree of injury or damage to the host. When the damage becomes pathogenic, intervention is required to save or minimize damage to the host.
In theory, diseases and disorders that should be most easily treated are those caused by organisms which are distinguishable from the host, precisely the situation found in parasitism. It should be possible to selectively kill or render the infecting organism nonpathogenic without destroying the host by taking advantage of a fundamental difference between the host and the parasite. Unfortunately, this is all too often not possible or unsuccessful because of an inability of the available drugs to destroy the organism at a concentration which is non-toxic to the host. This is especially true with regard to eukaryotic parasites.
Eukaryotic organisms which may be parasitic are varied and diverse. Some, such as Dracunculus medinensis, are over a meter in length whereas others, for example viruses, are microscopic. One of the largest groups of parasites are those which infect the gastrointestinal system. These include organisms which inhabit the mouth, stomach, small and large intestines, the upper respiratory passages and the urogenital tract. The list includes species of the genera Entamoeba such as Entamoeba histolytica, E. coli and E. poleki, flagellates such as Giardia lamblia, Dientamoeba fragilis and Trichomonas vaginalis, sporozoans such as Isospora belli and Toxoplasma gondii, sarcocysts and the ciliate Balantidium coli. It also comprises a number of commensals, species of questionable pathogenicity and certain opportunistic pathogens and commensals. These organisms have a world-wide distribution pattern with a prevalence that varies roughly with the measure of sanitation available in the area. Their degree of pathogenicity varies greatly both within and between the various genera and species.
Amebiasis has been implicated in dysenteric diseases, non-dysenteric colitis, acute non-suppurative hepatitis and liver and pulmonary abscesses. Treatments vary with the stage of the infection. The mainstays include iodo-hydroxyquinoline, diiodohydroxyquin (Diodoquin) and tetracycline. Additional compounds which have demonstrated utility include metronidazole (Flagyl) and diloxanide furoate (Furamide). Although most antibiotics are ineffective, intravenous amphotericin B, oral rifampin and certain combinations which appear to act synergistically may be used. Flagellates are treated with quinacrine hydrochloride, an acridine dye, or metronidazole (Flagyl). Ciliates such as Balantidium coli are treated with oxytetracycline (Terramycin) or diiodohydroxyquin (Diodoquin).
Another group of parasites that infects the gastrointestinal tract are the intestinal nematodes which is believed to encompass over 500,000 different species. These are the most worm-like of the parasitic animals with generally cylindrical shapes, tapered at both ends, and covered with a tough protective layer or cuticle. They have a complete digestive tract with both oral and anal openings on the separate sexes. Some nematodes are obligate parasites such as Wuchereria while others have a free-living period. Most are not long-lived and to persist in a host, must be continually reinfected.
The major intestinal round worm is Ascaris lumbricoides which ranges in size from 20 cm to 35 cm and is estimated to infect over 650 million people in the world today. Most of these infections are asymptomatic, but large numbers of parasite bodies can cause serious gastrointestinal disorders in heavy infestations. These worms can migrate to various organs and cavities within the body. The degree of pathogenic severity depends to a large extent on the area affected.
The pinworm, Enterobius vermicularis, is the most common helminth parasite on the temperate regions of the world. Spread is facilitated in crowded and unsanitary conditions. This organism is generally considered to be a commensal parasite. Symptoms range from mild irritation to sever inflammation, especially in those individuals who may be hypersensitivity to its secretions.
The hookworm occurs in both new and old world varieties. The new world hookworm, Necator americanus is found over the Western hemisphere and is still prevalent in certain areas of the United States. The old world hookworm, Ancylostoma duodenale, is found mainly in Europe and the Mediterranean and both varieties are prevalent in South America. Although most symptoms are fairly mild, such as itching and allergic reactions, there is a direct correlation between worm load and anemia. These parasites consume a substantial portion of the available iron from infected individuals and for those whose nutrition is already sub-adequate, severe complications including mental and physical retardation can result.
Other nematodes include Ancylostoma braziliense and A. caninum, the causative agents of cutaneous larva migrans, A. philippine the causative agent of philippine capillariasis which has a fairly high fatality rate, Strongyloides stercoralis which produces local lesions by penetrating the skin of human hosts, and the whipworm, Trichuris trichiura which has a worldwide distribution and a symptomology that directly correlates to infectious load. Some of the more problematic nematodes include those acquired from fish. This includes the roundworm larvae belonging to the genera Anisakis and Phocanema, and also related genera. Infections are reported from ingestion of raw, undercooked or even inadequately pickled fish. Diseases in humans are accompanied by a low grade eosinophilia and occult blood, but are generally not life threatening.
The most effective treatment presently available against the intestinal nematodes may be pyrantel pamoate (Antiminth) which has a cure rate of over 95% for ascaris (V. M. Villarejos et al., Am. J. Trop. Med. Hyg. 20:842-45, 1971). Other drugs which have proved to be useful include thiabendazole (Mintezol), pyrvinium pamoate, bephenium (Alcopara), mebendazole (Vermox), and combinations of these compounds administered either simultaneously or in a multi-course treatment regiment
The blood and tissue dwelling nematodes include the filariae which are long, thread-like nematodes that infect and reside in the blood and lymphatic systems, and also tissues such as the subcutaneous and deep connective tissues. These include Brugia malayi, B. timori, Loa loa (the African eye worm), Dipetalonema stretocerca and Onchocerca volvulus. Bancroftian filaria are widely distributed throughout the tropics and subtropics. One characteristic representative of this group is the parasite Wuchereria bancrofti which is transmitted by mosquitoes. Within the infected mosquito, microfilaria undergo an essential developmental cycle transforming into infective larva. These larvae then enter the proboscis of the mosquito and, at the mosquito's next blood meal, leave the proboscis and enter the host through the puncture hole left by the mosquito. Clinical manifestations vary and appear to depend upon the numbers of infecting organisms and the present physical state of the host. Early symptoms of filariasis are fever, lymphangitis and lymphadenitis. Attacks are sometimes referred to as elephantoid fever with an associated lymphangitis most commonly affected the lymph nodes. Elephantiasis, the enlargement of one or more limbs, scrotum, breasts or vulva, is relatively uncommon and a late complication of filariasis. It is caused by obstruction of lymph flow and typically accompanied with multiple abscesses on the body. Obstruction is not, as is often believed, caused by massive numbers of living or dead filariae occluding vessels, but is more likely caused by an allergic reaction to the filaria in the tissues which surrounds the vessels. Individuals with especially severe infectious loads have multiple complications.
Treatment is usually an antihistaminic such as promethazine hydrochloride co-administered with steroids to reduce inflammation. The standard filaricide is diethylcarbamazine and its derivatives which is often followed with a regiment of suramin. These treatments often require months of administration, a significant problem in most ares of the world.
Another common tissue dwelling nematode is Trichinella spiralis. In intestinal infections, the worm is found in the gut mucosa and usually asymptomatic. Upon entry into a migratory phase, these parasites travel through the body lodging in muscles forming encased larval cysts that can produce severe symptoms. Muscle cysts can cause hemorrhages, edema, visual disturbances and pain. Central nervous system involvement is common in more advanced stages and can lead to death.
Treatment of symptomatic infections usually involves corticosteroids such as prednisone or prednisone derivatives. Thiabendazole may be somewhat effective, but its utility has not been proven. Mebendazole is somewhat effective in preventing tissue infection and may also be useful against the tissue phase as well.
Tissue coccidia are intestinal parasites of man with a more cosmopolitan distribution. Toxoplasma gondii infects a variety of vertebrates including cats which can transfer the disease to man. Most infections are benign, however, infections of cardiac patients and infections during pregnancy can lead to serious complications. The only treatment available is long term administration of pyrimethamine with supplements of folic add to counter expected suppression of bone marrow cell proliferation.
Sarcocystis, babesia and pneumocystis, three additional forms of tissue coccidia, are intestinal parasites of man and domesticated and wild animals. Infants and young children are especially sensitive to these diseases. Treatment usually involves combinations of sulfadiazine, pyrimethamine and leucovorin, or sulfamethoxazole and famethoxazole. Side effects, some of which can be severe, are common.
Another tissue parasite, common in the areas from Asia to the Arabian peninsula and from Africa to southern Russia is Dracunculus medinensis, the guinea worm. These worms develop in water as copepods and are ingested or infected through the skin. They live and mature in the deep connective tissues of the body for approximately one year. Mature worms migrate to a position close to the skin forming ulcerations. Upon contact with water, the female worms discharge large numbers of copepods into the water to repeat the cycle. Worms generally must be removed surgically which is an extremely difficult procedure due to the fragility of the worm's body which if ruptured floods the patient's body with toxins and extremely allergenic substances. Secondary infections are common and represent just one of the more persistent problems associated with this infection. Treatment typically involves oral administration of niridazole (Ambilhar) or metronidazole. These compounds tend to produce tolerable side effects including nausea, vomiting and diarrhea, and also more severe effects such as anemia, paresthesia and electrocardiographic changes.
Among the most important of the blood and tissue dwelling protozoa are the hemoflagellates. The principle families of this group are the Trypanosomidae and Leishmania, both of which are a significant source of morbidity and mortality in the world today.
Trypanosomiasis occurs in many different forms in man. African sleeping sickness, caused by Trypanosoma brucei gambiense, the more virulent T. brucei rhodesiense, and T. brucei brucei which is restricted to animal hosts, are nearly impossible to distinguish morphologically. All are transmitted by the bite of the tse tse fly (Glossina palpalis). Typically, an ulceration will appear in the area of the bite (trypanosomal chancre) that slowly disappears. Incubation periods range from days to weeks during which time the patient is symptomless although trypanosomes can be found in the blood. Infections can be abortive or invade the lymphatic tissues which is signaled by the onset of rigor and febrile attacks lasting for weeks or even months. Malaise and headache usually accompany an attack along with night sweats and an overall anorexia. Often nodes become visible (Winterbottom's sign) and there is an increasing lassitude and apathy in the patient as the disease progresses to central nervous system (CNS) involvement. As mental faculties deteriorate, there is a general fatigue, confusion and somnolence. Extreme emaciation is observed in patients who have received little or no medical attention. Motor function becomes increasingly difficult with an evident slurring of speech and an ataxic gait. Pressure on the palms produces severe pain after the pressure is removed (Kerandel's sign), a signature characteristic of the disease.
In the final stages, there are profound character changes and mental deterioration accompanied with convulsions, hemiplegia and paraplegia, incontinence and severe paresthesia until the patient becomes comatose. Relapses and remissions are common and can continue for years until the patient eventually succumbs. Treatment is typically melarsoprol, Mel B or melarsen oixide complexed with dimercaprol. Suramin is generally effective, but additional drugs which can be used include pentamide and derivatives of melarsoprol.
American trypanosomiasis (Chagas disease) is caused by Trypanosoma cruzi and occurs throughout the southern parts of North America and northern parts of South American. These parasites are transmitted from the bite of the reduviid bug (Panstrongylus megistus). The disease is sever in children under five where central nervous system involvement predominates. The site of the infection occurs most frequently on the face creating an intense inflammatory reaction or a chagoma (Roman's sign). Symptoms appear in about four to fourteen days after the bite. Promastigotes migrate to the lymph system and quickly lodge to regional lymph nodes where they are ingested by cells in the node. Within these cells, promastigotes transform into amastigotes which can migrate throughout the body and lodge within the tissues of any organ.
In adults there are few symptoms, but in children there can be chills, high fevers, muscular aches, an increasing exhaustion and epitaxis. Liver, spleen and cardiac cells tend to be favorite sites of infection. Chronic infections are accompanied by heart problems such as massive cardiomegaly and fibrosis. The only effective treatment is nitrofurfurylidine derivative Bayer 2502 (Nifurtimox). It is most useful in early stage and acute stages of the disease and requires extended treatment periods.
Another major hemoflagellate disease is leishmania which can be produced from a variety of species of Leishmania, all of which are transmitted by the sandfly (Phlebotomus). Cutaneous leishmaniasis, the oriental sore, is caused by Leishmania tropica found throughout Asia Europe and Africa, and L. mexicana which is endemic to the Americas. Incubation periods after an initial infection may require months or even years. During incubation there are few, if any, symptoms. Small red papule may appear on the skin at the site of infection which will itch and form open ulcerations with a serious exudate. Most lesions heal spontaneously as a natural immunity develops. Exposure confers absolute resistance to reinfection by the same species.
Mucocutaneous leishmardasis (L. braziliensis) and visceral leishmaniasis or kala-azar (L. donovani), are transmitted by sandflys. As their names implies, these diseases affect specific tissues of the patient. Symptoms include ulcerations of the soft tissues and in the more serious cases, complete destruction and loss of cartilaginous tissues. Visceral leishmaniasis can occur at any area of the body and is generally preceded by a darkening at the site of infection and a parasitemia of the reticuloendothelial system throughout the body. Mucocutaneous leishmaniasis is more restricted to the mucosal tissues where secondary infections become common and prominent.
Treatment for all forms of leishmaniasis requires administration of sodium gluconate (Pentostam) which is considerably less toxic than most antimoniuals. Side-effects with these drugs are common.
One of the most well-known parasitimias in the world today is malaria. Malaria can present itself as several different clinical syndromes which are now known to be caused by several different species of Plasmodium including P. falciparum, P. vivax, P. ovale and P. malariae. Both morphologically and clinically, each can be easily distinguished (Table 1).
TABLE 1 ______________________________________ Comparison of the Clinical Course of Various Forms of Plasmodium. P. P. P. vivax P. ovale malariae falciparum ______________________________________ Incubation Time (days) 10-17 10-17 18-40 8-11 Severity severe mild severe mild Fever pattern irregular irregular regular continuous quotidian quotidian 72 hours quotidian Periodicity (hours) 48 48 72 36-48 Untreated Duration 5-7 years 12 months 20+ years 6-17 months Anemia ++ + ++ ++++ CNS Involvement + +/- + ++++ Nephrotic Syndrome +/- - +++ + ______________________________________
Human malaria was first recognized in the late 1800's and by the early 1900's, the entire life cycle of the parasite from man to mosquito was known. During this period, it was also discovered that Plasmodium occurs in others species of mammals as well as man. The life cycle of the parasite begins when the female mosquito bites an infected person and ingests infected blood containing male and female gametocytes. In the mosquito, the male gametocyte undergoes a process of maturation, termed exflagellation, extruding delicate spindle-shaped gametes. Simultaneously, the female gametes also mature and become fertilized by the male gametes forming zygotes. These zygotes elongate into ookinetes which penetrate the stomach wall of the mosquito lodging within the outer covering of cells to become oocysts. The oocyst develops into thread-like sporozoites that break out and wander throughout the mosquito's body, eventually finding their way into the salivary glands to be introduced into the blood stream of the mosquito's next host.
Once injected the sporozoites leave the blood vascular system within about forty minutes and invade the parenchymal cells of the liver. In the liver, the parasites undergo a period of asexual development termed the pre-erythrocytic stage. After maturation, parasites are liberated into the blood stream to infect red blood cells. Another stage of asexual development occurs in red blood cells in a process termed schizogony. Within a period of about 72 hours, handfuls of new parasites form from each infected cell. Details of this cycle differ from species to species. At the end of this period, red blood cells rupture liberating more merozoites which in turn infect more cells. It has been suggested that fever induced by the near simultaneous rupture of large numbers of red blood cells dumping large amounts of toxins into the blood stream has some regulatory effect on the developmental cycle. Febrile periodicity becomes synchronized at 48-72 hour intervals depending on the species.
At some time after asexual development, gametocytes appear in red blood cells and the patient begins to show pathologic signs of infection. These parasitic forms continue to grow and develop asexually, but do not divide and eventually form the male and female forms. It is these forms which are ingested by the mosquito to begin the cycle again.
Treatment regiments for malaria are quite varied. Colchicine, sulfones such as diaminodiphenylsulfone (dapsone), and the longer-acting sulfonamindes are PABA antagonists interfering with the synthesis of folic acid from para-aminobenzoic acid which effects the schizont stage of the erythrocytic life cycle. Chlorguanide (Paludrine), a biguanide, and the structurally related drugs diaminopyrimidine and pyrimethamine (Daraprim), are relatively slow-acting anti-malarials that effect the sexual erythrocytic stages of malaria by interfering with the metabolism of folic acid.
The three separate stages of the plasmodial life cycle in man cannot be successfully treated with any single drug. The pre-erythrocytic and erythrocytic cycles are sensitive to primaquine and also to chlorguanide and pyrimethamine. The sexual stages respond to quinine, chloroquine, hydroxychloroquine, amodiaquine and quinacrine, chloroquine and pyrimethamine, sulfonamides and sulfones, and colchicine. Combination therapies are designed based on these various modes of action. Chemoprophylaxis is usually performed for those who must travel into endemic areas and for those uninfected individuals who may require extra measures of protection. The drugs of choice for prophylaxis are chloroquine and hydroxychloroquine. All of these drugs have been used extensively in areas of the world in which malaria is endemic. Unfortunately, significant resistance has also developed in the various species as well.
Others parasitemias of man include the trematodes such as the liver flukes (Fasciola hepatica, Opisthorchis and Dicrocoelium), the intestinal flukes (Fasciolopsis buski, the Echinostomes and the Heterophyids), and the liver flukes (Paragonimus westermani and P. heterotremus). These organism are extremely varied with complex life cycles. Lung flukes are treated with bithionol which is widely used in medicated shampoos and soaps. Intestinal flukes are treated with niclosamide (Yomesan) which can be absorbed through the gastrointestinal tract. Praziquantel is the treatment of choice for liver flukes.
The blood flukes, Schistosoma mansoni, S. japonicum and S. haematobium, are of particular importance because of their widespread distribution. These parasites cause a very large portion of morbidity and mortality in the world today. Schistosomes have a complex life cycle which generally begins in eggs that find their way into bodies of water from contaminated feces. Once in contact with water the eggs hatch into miracidium which infect the snail. Within the snail, larval stages develop giving rise to large numbers of cercariae that are subsequently liberated into the water. Humans infection occurs upon contact with cercariae infested water as the parasites penetrate directly through the skin. After penetration, parasites invade blood vessels and are carried to the lungs and than the liver where they begin their growth. About two weeks later, adult worms begin a migration against the flow of blood and into the portal system lodging in the mesenteric or vascular veins. Final locations differ for each species. Eggs are produced which travel throughout the body. Some travel to the intestines to be excreted to begin the cycle again while others lodge in various parts of the body.
Upon penetration of the skin there is sometimes a transient reaction such as edema and puritis, but little more. During incubation there may be allergic reactions and a generalized malaise or fever. When the flukes reach their final location, the acute stage of the disease begins and symptoms vary from mild to severe. There is usually some intestinal and bladder pain and tenderness, diarrhea, fever, dysuria and hematuria accompanied by eosinophilia. In the third stage the disease become chronic and many of the complications associated with third stage schistosomiasis can be attributed to the eggs. There is extensive fibrosis and hyperplasia of the tissues where eggs are deposited. Abscesses form with severe hepatomegaly and splenomegaly. In addition, the loss of red blood cells is severe. Adult females have been demonstrated to consume as many as 300,000 red blood cells per hour and adult males about 30,000. In areas where individuals are likely to be infected, nutrition is already quite poor complicating treatment and any good prognosis for recovery.
Blood flukes are treated with potassium antimony tartrate or tartar emetic, a trivalent antimony compound which for a long time was the treatment of choice. Stibophen and oxamniquine are used and have demonstrated some effectiveness. Antimonials are contra-indicated in the presence of cardiac or renal disease. Oxamniquin (Vansil) is the drug of choice with S. mansoni and metrifonate (Bilarcil) for S. haematobium. Niridazole (Ambilhar) is acceptable for most infections and is typically effective against S. japonicum. Other compounds which may be useful include amoscanate, praziquantel and sodium antimony dimercaptosuccinate (Astiban).
Although there has been some success with current treatments of parasitic infections, these diseases remain serious sources of morbidity and mortality in the world.